Transglutaminases (TGases; protein-glutamine: amine {gamma}-glutamyl-transferases) are a diverse family of Ca+2 dependent enzymes with distinct genes, structures and biological functions. They are responsible for blood clotting, apoptosis, seminal fluid coagulation, extracellular matrix, bone formation, and barrier formation in stratified squamous epithelia. The common reaction performed by each of the nine TGase enzyme isoforms known in the human genome involves the activation of a target or donor protein-bound gamma carboxyamide group of a glutamine residue and an acceptor nucleophile of which four classes are known. If the nucleophile is: (i) a polyamine (such as spermidine), a mono- or bis-(gamma-glutamyl)spermidine linkage is formed; (ii) the epsilon-NH2 group of a protein-bound lysine residue, an isopeptide N-epsilon-(gamma-glutamyl)lysine cross-link is formed; (iii) water, the net result is deamidation of the glutamine (to glutamic acid); or (iv) the terminal {omega}-alcohol group of certain long-chain epidermal-specific ceramides, a glutamate ester linkage results. Among the nine TGase enzyme isoforms known in the human genome, only TGase 2 wasknown to bind and hydrolyze GTP/GDP; binding GTP inhibits its transamidation activity but allows it to function in signal transduction. We presented biochemical and crystallographic evidence for the direct binding of GTP/GDP to the active TGase 3 enzyme, and showed that the TGase 3 enzyme undergoes a GTPase cycle. The crystal structures of active TGase 3 with GTP{gamma}S and GDP were determined to 2.1? and 1.9? resolution, respectively. These studies reveal for the first time the reciprocal actions of Ca2+ and GTP with respect to TGase 3 activity. GTP{gamma}S binding results in the replacement of a bound Ca2+ with Mg2+, and conformational rearrangements that together close a central channel to the active site. Hydrolysis of GTP to GDP results in two stable conformations, resembling both the GTP state and the non-nucleotide bound state, the latter of which allows substrate access to the active site. We also solved the crystal structure determined at 2.0 ? resolution of TGase 3 in complex with GMP in order to elucidate the structural features required for nucleotide recognition. Binding affinities for various nucleotides were found by fluorescence displacement to be as follows: GTP{gamma}S (0.4 uM), GTP (0.6 uM), GDP (1.0 uM), GMP (0.4 uM), and ATP (28.0 uM). Furthermore, we found that GMP binds as a reversible, noncompetitive inhibitor of TGase 3 transamidation activity, similar to GTP{gamma}S and GDP. A genetic algorithm similarity program (GASP) approach (virtual ligand screening) identified three compounds from the Lead Quest TM database (Tripos Assoc. Inc) based on superimposition of GTP{gamma}S, GDP, and GMP guanine nucleotides from our crystal structures to generate the minimum align flexible fragment. These three were nucleotide analogs without a phosphate group, containing the minimal binding motif for TGase 3 that includes a nucleoside recognition groove. Binding affinities were measured as follows: TP349915 (Kd=4.1uM), TP395289 (Kd=38.5 uM), TP394305 (Kd=1.0 mM). Remarkably, these compounds, do not inhibit, but instead activate TGase 3 transamidation by about 10-fold. These results suggest that the nucleotide-binding pocket in TGase 3 may be exploited to either enhance or inhibit the enzymatic activity as required for different therapeutic approaches. These structures, backed with extensive biochemical studies, are providing new insights as to how access to the enzyme?s active site may be gated through the coordinated changes in cellular calcium and magnesium concentrations and GTP/GDP. Calcium-activated TGase 3 can bind, hydrolyze, and is inhibited by GTP, despite lacking structural homology with other GTP binding proteins. A structure based sequence homology among the TGase enzyme family shows that these essential structural features are shared among other members of the TGase family. The human S100A15 is a novel member of the S100 family of EF-hand calcium binding proteins that is recognized to be TGase 3 substrate. We are currently in the process of crystallizing the TGase 3 with human S100A15. The human S100A15 was recently identified in psoriasis, where it is significantly upregulated in lesional skin. The protein is implicated as an effector in calcium-mediated signal transduction pathways. Although its biological function is unclear, the association of the 11.2 kDa S100A15 with psoriasis suggests that it contributes to the pathogenesis of the disease and could provide a molecular target for therapy. To provide insight into the function of S100A15, we have crystallized the protein to visualize its structure and to further the understanding of how the many similar calcium-binding mediator proteins in the cell distinguish their cognate target molecules. We have cloned, expressed and purified the S100A15 protein to homogeneity and produced two crystal forms. The crystals of form I are triclinic with cell parameter a = 33.5 ?, b = 44.3 ?, c = 44.8 ?, {alpha} = 71.2?, {beta} = 68.1?, and {gamma} = 67.8?, an estimated two molecules in the asymmetric unit and diffraction to 1.7 ? resolution. The crystals of form II are monoclinic (space group C2) with cell parameter a = 82.1 ?, b = 33.6 ?, c = 52.2 ? and {beta} = 128.2?, an estimated one molecule in the asymmetric unit and diffraction to 2.0 ? resolution. This structural analysis of the human S100A15 will further aid in the phylogenic comparison between the other members of the S100 protein family, especially the highly homologous paralog S100A7. Another ongoing project involves crystallizing TGase 3 with cystamine. At least three types of TGases (1, 2, and 3) are expressed in human brain which are responsible for the TGase activity that produces cross-linked protein or amine conjugate. It has been suggested that TGases play a critical role in the common pathogenesis of CAG trinucleotide-repeat neurodegenerative diseases. TGase catalyzed aggregates are formed when the polyglutamine domain of huntingtin (HD) exceeds a stretch of 36 expanded glutamines becoming cross-linked with polypeptides containing lysyl groups to form covalent bonds. This action is due to increased TGase activity in the HD brain. Cystamine (beta, beta'-diaminodiethyl disulfide), a thiolamine joined by a disulfide bridge between two cysteamine (beta-mercaptoethylamine) is shown to be neuroprotective in a number of disease models. It also has been demonstrated that cystamine reduces brain TGase activity as well as TGase-positive aggregate-like figures that colocalize with HD aggregates. In addition, there is evidence that both free and protein-bound ({gamma}-glutamyl)lysine levels are reduced/normalized in HD mice following cystamine administration. Consequently, there is a great interest in both the therapeutic potential of this agent and its mechanism of action, which includes the inhibition of TGase activity. It is not yet clear if cystamine?s mechanism of action is competition for the substrate of the TGase enzyme or whether it directly inhibits TGase activity.